Method of treating or preventing type 1 diabetes by oral administration of insulin

ABSTRACT

Disclosed herein are methods for treating or preventing a disease in mammals having the characteristics of Type 1 diabetes comprising administering insulin or disease suppressive fragments of insulin or analogs thereof in oral or aerosol dosage forms to said mammals. Also disclosed herein are pharmaceutical formulation or dosage forms for use in the methods.

This is a continuation of application Ser. No. 08/235,121, filed Apr.28, 1994, now abandoned, which is a continuation of 08/070,020 filed May28, 1993, now abandoned, which is a continuation of 07/896,484 filedJun. 02, 1992, now abandoned, which is a continuation of 07/595,468filed Oct. 19, 1990 now abandoned.

FIELD OF THE INVENTION

This invention relates to methods and compositions for treating orpreventing Type 1 diabetes mellitus. The methods of the invention do notrequire parenteral administration of therapeutic agents. Moreparticularly, but not by way of limitation, the present invention isdirected to agents and methods for treating or preventing autoimmunediseases having the characteristics of Type 1 diabetes mellitus.

BACKGROUND OF THE INVENTION

Diabetes mellitus is characterized in two broad groups based on clinicalmanifestations, namely, the non-insulin-dependent or maturity onsetform, also known as Type 2; and the insulin-dependent or juvenile onsetform, also known as Type 1. Clinically, the majority of Type 2, maturityonset diabetics are obese, with manifestations of clinical symptoms ofthe disease usually appearing at an age over 40. In contrast, Type 1,juvenile onset patients are not over-weight relative to their age andheight, with rapid onset of the disease at an early age, often before30, although Type 1 diabetes can occur at any age.

Diabetes mellitus is a metabolic disorder in humans with a prevalence ofapproximately one percent in the general population, with one-fourth ofthese being the Type 1, insulin-dependant category (Foster, D. W.,Harrison's Principles of Internal Medicine, Chap. 114, pp. 661-678, 10thEd., McGraw-Hill, New York). The disease manifests itself as a series ofhormone-induced metabolic abnormalities which eventually lead toserious, long-term and debilitating complications involving severalorgan systems including the eyes, kidneys, nerves, and blood vessels.Pathologically, the disease is characterized by lesions of the basementmembranes, demonstrable under electron microscopy.

Type 1 diabetics characteristically show very low or immeasurable plasmainsulin with elevated glucagon. Regardless of what the exact etiologyis, most Type 1 patients have circulating antibodies directed againsttheir own pancreatic cells including antibodies to insulin, to islet ofLangerhans cell cytoplasm and to the enzyme glutamic acid decarboxylase.An immune response specifically directed against beta cells (insulinproducing cells) leads to Type 1 diabetes. This specificity is supportedby the above clinical picture, since beta cells secrete insulin whilealpha cells secrete glucagon.

Current therapeutic regimens for Type 1 diabetes include modificationsto the diet in order to minimize hyperglycemia resulting from the lackof natural insulin, which in turn, is the result of damaged beta cells.Diet is also modified with regard to insulin administration to counterthe hypoglycemic effects of the hormone. Whatever the form of treatment,parenteral administration of insulin is required for all Type 1diabetics, hence the term "insulin-dependent" diabetes.

Conventional insulin therapy is limited to the parenteral (i.e.subcutaneous) administration of insulin. Oral administration of insulinhas not been possible because the insulin molecule cannot pass throughthe digestive tract in a sufficiently intact form to provide itstherapeutic benefit. There has been an ongoing search by those skilledin the art for an alternative method to eliminate or reduce the need forinsulin because of the numerous problems associated with subcutaneousadministration of the drug. Among the methods that have beeninvestigated are implantable insulin pumps and pancreatic islet celltransplantation.

Because Type 1 diabetes usually manifests itself in adolescents andbecause the subcutaneous delivery of insulin requires strict,self-regimentation, compliance is often a serious problem. In addition,the act of the parenteral administration itself can be very traumatic tojuveniles. For the clinician, it is difficult to precisely regulate theamounts of insulin needed at any given time of the patient's day.Furthermore, it is all but impossible to regulate blood glucose levelsin diabetic patients with parenteral insulin to the extent to whichblood glucose is regulated in normal individuals.

Thus, in the early stages of treatment of Type 1 diabetes, patientsoften become either hyperglycemia or hypoglycemic because the exacttiming of the insulin injections and levels of insulin needed are notknown. As treatment progresses the clinician and, more importantly, thepatient adjusts to the daily routine, but there is always the risk ofketoacidosis or hypoglycemia.

In addition, some patients produce antibodies to the injected insulineven though most patients are now treated with human insulin produced byrecombinant technology. This can lead to the need for higher doses ofinsulin. To date, there has been no successful oral dosage formcontaining insulin.

The art has long sought a method for preventing and/or treating Type 1diabetes which does not involve parenteral administration of insulin.Various attempts at alternate delivery methods for insulin have failedor have not progressed to a practical level. For example, oral insulin,transcutaneous insulin delivery, and nasal insulin have not becomeclinically utilized. Oral insulin does not affect blood glucose levels.Because it has been necessary to inject lipids into mammals receivingnasal insulin in order to get the insulin across the nasal mucosa, thisroute of insulin administration has been limited.

Oral dosage forms for treatment of Type 2 diabetes are available, (e.g.oral sulfonylureas). These orally administered agents do not compriseinsulin but rather stimulate the pancreas to produce insulin, and areonly effective for treatment of certain forms of Type 2 diabetes. As forType 1 diabetes, oral agents such as sulfonylureas are ineffective dueto markedly diminished or damaged beta-cell mass (Foster, D. W.,Harrison's Principles of Internal Medicine, Chap. 114, p. 668, 10th Ed.,McGraw-Hill, New York).

Type 1 diabetes is considered to be a disease of autoimmune etiology(Eisenbarth, G. S., New Engl. J. Med. 314: 1360-1368, 1986). Variousanimal models are available for the study of Type 1 diabetes as anautoimmune disorder. These include the BB mouse (Nakbookda, A. F., etal., Diabetologic 14: 199-207, 1978) and the NOD (non-obese diabetic)mouse in which diabetes develops spontaneously (Prochazka et al. Science237:286, 1987). Islet-cell specific, CD4 and CD8 T-lymphocytes have beenimplicated as causative agents for damage to beta cells, demonstrated bydecreased incidence of Type 1 diabetes in NOD mice (J. Exp. Med.166:823, 1987).

Other therapies are being developed for the treatment of autoimmunediseases in general.

Weiner et al., U.S. patent application Ser. No. 460,852 filed Feb. 21,1990, now abandoned (the national stage of PCT Application No.PCT/US88/02139, filed Jun. 24, 1988), which is a continuation-in-partapplication of U.S. patent application Ser. No. 065,734 filed Jun. 24,1987, now abandoned discloses the treatment of autoimmune diseases byoral administration of autoantigens.

Weiner et al., U.S. patent application Ser. No. 454,806 filed Dec. 20,1989, now abandoned discloses the aerosol administration ofautoantigens, disease-suppressive fragments of said autoantigens andanalogs thereof as an effective method for treating T-cell mediatedautoimmune diseases.

Weiner et al., U.S. patent application Ser. No. 487,732, filed Mar. 2,1990, now abandoned discloses synergists (enhancers) for use with oraladministration of autoantigens, disease suppressive fragments andanalogs thereof as effective treatments for T-cell mediated autoimmunediseases.

Weiner et al., U.S. patent application Ser. No. 551,632 filed Jul. 10,1990, now abandoned as a Rule 62 continuation-in-part application ofU.S. patent application Serial No. 379,778, filed Jul. 14, 1989 (nowabandoned), discloses methods of preventing or treating uveoretinitis inmammals by oral administration of purified S antigen.

Nagler-Anderson, et al., (Proc. Natl. Acad. Sci (USA) 83: 7443-7446,1986), describe the oral administration of collagen to suppresscollagen-induced arthritis in a mouse model.

However, none of the above-mentioned for treatments for autoimmunediseases can be used treating Type 1 diabetes because the antigensinvolved in eliciting and maintaining the Type 1 disease have not beenidentified.

It is, therefore, an object of the present invention to provide agentsand methods for treating and preventing autoimmune diseases in mammalshaving the characteristics of Type 1 diabetes. Prevention of Type 1diabetes preferably involves treatment to hyperglycemia.

Another object of the present invention is to provide compositions andpharmaceutical formulations useful for treating mammals suffering fromautoimmune diseases having the characteristic of Type 1 diabetes.

A still further object of the invention is to provide compositions andpharmaceutical formulations useful for oral administration to mammalsfor the purpose of preventing or attenuating the manifestations (i.e.clinical symptoms) of autoimmune diseases having the characteristics ofType 1 diabetes. Prevention of Type 1 diabetes includes treatment priorto hyperglycemia.

These and other objects of the present invention will be apparent tothose of ordinary skill in the art in light of the following.

SUMMARY OF THE INVENTION

It has unexpectedly been discovered that oral administration of insulinis an effective treatment for eliminating or reducing the need forinsulin in Type 1 diabetics. Oral insulin can prevent or ameliorate betacell distruction and thereby decrease or eliminate traditionalparenteral insulin therapy.

Orally administerable pharmaceutical formulations containing insulin areprepared and administered to mammals who have manifested symptoms ofType 1 diabetes and/or diagnosed as having Type 1 diabetes.Additionally, subjects who are at risk for developing Type 1 diabetes(i.e. have demonstrated a predisposition to developing Type 1 diabetesthrough suitable means, such as genetic studies and analysis) aretreated with similar oral preparations of insulin.

Pharmaceutical formulations for oral or enteral administration to treatType 1 diabetes are prepared from commercially available insulin and apharmaceutically acceptable carrier suitable for oral ingestion. Thequantity of insulin in each dose may be between 1 mg and 1000 mg.However the total dose required for treatment varies according to theindividual. Generally, the total quantity of insulin required inpracticing the present invention is a much larger dose than is thedosage that is administered parenterally to protect an individualafflicted with Type 1 diabetes against ketoacidosis.

Additionally, an aerosol delivery system can be made with similardosages of insulin as above with a pharmaceutically suitable carrier ordiluent. These and other improvements will be described in the followingdescriptions, drawings and the appended claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a graph showing the effects of orally administered porcineinsulin on diabetes in NOD mice.

FIG. 2 is a graph showing the effects of oral porcine insulin on serumglucose concentrations post-administration of oral insulin in NOD mice.

FIG. 3 is a graph showing the effect of oral administration ofpancreatic extracts given at weekly intervals on the development ofdiabetes in NOD mice.

DETAILED DESCRIPTION OF THE INVENTION

All patent applications, patents and literature references referred toherein are hereby incorporated by reference in their entirety.

The present invention addresses the need for an alternate to existingmethods for the treatment of Type 1 diabetes from an autoimmune modeland in particular at treatment for the prevention of diabetes. BecauseType 1 patients are predominantly adolescents, it is believed that longterm insulin therapy will no longer be required due to preservation ofundamaged beta cell function if treatment is initiated at the onset ofthe disorder using the compositions and methods of the presentinvention. In addition, if the methods of the present invention areinitiated at a time when some beta cell function still exists,parenteral insulin therapy can be reduced. Thus, the present inventionprovides means whereby long term insulin therapy would be reduced or nolonger be required.

It has now been unexpectedly discovered that oral and/or aerosoladministration of insulin (or disease-suppressive fragments of insulinor analogs thereof) is effective for the treatment and prevention ofType 1 diabetes. This is a radical departure from traditional(parenteral) insulin therapy in that insulin is administered in order todamp down or shut off the hosts autoimmune response and not for itsendocrinologic (metabolic) effect. Oral administration of insulin hasnot proved to be effective in treating any form of diabetes becauseproteolytic enzymes present in the stomach and digestive system degradethe polypeptide before it can reach the bloodstream. In addition,intranasal administration of insulin has also not previously been shownto be effective in treating any form of diabetes.

Without wishing to be bound by theory of operation it is believed thatthe oral or aerosol administration of insulin pursuant to the presentinvention affects the immunological pathogenesis of Type 1 diabetesthrough the elicitation of suppressor T-cells.

In the following discussions the following terms shall have the meaningascribed to them below.

"Treatment" shall mean the treatment of active disease in patients withsome level of undamaged, insulin-producing beta cells as well asprophylactic administration for use in patients having a high risk fordeveloping the disease.

"Oral administration" shall mean both oral administration and enteraladministration (direct incubation into the stomach).

"Individuals at risk" for Type 1 diabetes shall mean a) individualshaving a blood relative with Type 1 diabetes; b) autoantibody-positiveindividuals without overt Type 1 diabetes. These auto antibodies includecytoplasmic islet cell autoantibodies, insulin antibodies and glutamicacid decarboxylase autoantibodies; c) individuals withHistocompatibility (HLA) type DR3 or DR4DQW8; d) individuals withglucose abnormalities such as a loss of first phase insulin secretion onglucose tolerance tests.

"Mammal" shall mean any organism having an immune system and thereforesusceptible to Type 1 diabetes.

"Active disease", shall mean autoimmune destruction of islet beta cells.

"Aerosol" refers to finely divided solid or liquid particles that may becreated using a pressurized system such as a nebulizer. The liquid orsolid source material contains insulin and/or disease suppressivefragments of insulin and analogs thereof as defined herein.

"Disease suppressive fragments" of insulin includes any peptide orpolypeptide containing partial amino acid sequences or moieties ofinsulin and possessing the ability to treat or prevent a disease havingthe characteristics of Type 1 diabetes. Such fragments need not possessthe autoantigenic or endocrinologic (metabolic) properties of the entireinsulin molecule.

"Analogs" of insulin or disease suppressive fragments thereof refers tocompounds that are structurally related to insulin or diseasesuppressive fragments thereof which possess the same biologic activity,i.e., the ability to suppress or prevent disease symptoms of Type 1diabetes, upon oral or aerosol administration. By way of non-limitingexample, the term includes peptides having amino acid sequences whichdiffer from the amino acid sequence of insulin or disease suppressivefragments thereof by one or more amino acid residues while stillretaining the disease suppressive activity of insulin or its ability toprevent or alleviate the symptoms of Type 1 diabetes. These analogs donot necessarily need to possess the endocrinologic effects of insulin.

In accordance with the present invention, experiments were performed inwhich NOD (non-obese diabetic) mice, which develop diabetesspontaneously between 11 and 52 weeks of age, had a lower incidence ofdiabetes at all doses of orally administered insulin in a dose-dependentmanner. In all of the animals which received the highest dose of oralinsulin (1 mg), 100% of the treated animals failed to develop diabetes.

Although it is fairly easy to control the symptoms of Type 1 diabeteswith parenteral insulin, it is difficult to normalize a patient's bloodsugar throughout 24 hours utilizing traditional insulin therapy given as1 or 2 injections a day. Thus, the present invention provides a methodfor treating or preventing a disease having the characteristics of Type1 diabetes in a mammal comprising administering to a mammal, sufferingfrom or at risk for Type 1 diabetes, an effective amount of an oralinsulin dosage form to treat or prevent the symptoms of diabetes.

It should be noted that the methods of the present invention will noteliminate the need for parenteral insulin therapy in patients withdamaged beta cells who do not produce enough insulin to regulate theirblood sugar. However, using the methods and compositions of the presentinvention, newly diagnosed Type 1 diabetic patients or those at risk forthe disease (as defined above), who have substantially intact(undamaged) beta cells, will not go on to develop Type 1 diabetes andparenteral insulin administration may be eliminated. In addition, it isbelieved that the methods and compositions of the present invention willreduce the amount of insulin needed by those individuals having somebeta cell function (who are able to produce some level of insulin) byhalting progression of the disease.

Insulin for use in the present invention can be obtained from numerouscommercial sources such as Novo Laboratories (Danbury, Conn.),Nordisk-USA (Rockville, Md.) and Eli Lilly and Co. (Indianapolis, Ind.).Porcine-derived insulin, human semi-synthetic insulin (Nordisk-USA) andcloned recombinant insulin (Eli Lilly) can be used when practicing themethod of the present invention.

Disease suppressive fragments and analogs of insulin for use in thepresent invention can be synthesized using well known solid phasesynthesis techniques (Merrifield, R. B. Fed. Proc. Am. Soc. Ex. Biol.21: 412, 1962 and J. Am. Chem. Soc. 85: 2149, 1963; Mitchel, A. R. etal., J. Am. Chem. Soc. 98: 7357, 1976; Tam, J. et al., J. Am. Chem. Soc.105: 6442, 1983). Analogs can be constructed by identifying anequivalent amino acid sequence and using the peptide synthesistechniques disclosed above.

Analogs can be provided using the known amino acid sequence of insulinas disclosed in Atlas of Protein Sequence and Structure, Natl. Biochem.Res. Foundation, vol. 5, pgs. 209-211.

Disease-suppressive analogs and fragments can also be obtained usingrecombinant DNA techniques well-known in the art.

Disease suppressive fragments of insulin and analogs thereof can beidentified using routine experimentation using suitable in vivo systemssuch as those of Examples 1-5 below.

Pursuant to the present invention, insulin or disease suppressivefragments or analogs thereof are introduced into a mammal suffering fromor at risk for a disease having the characteristics of Type 1 diabetes,orally or enterally, in an amount of between about 2 mg per kg bodyweight of said mammal and about 10 mg per kg body weight of said mammalper day, and may be administered in a single dose form or multiple doseforms. Preferably, the insulin is administered in an amount betweenabout 2.5 mg and about 5.0 mg per kg body weight of said mammal per day.The exact amount to be administered will vary depending on the severityand stage of a patient's disease and the physical condition of thepatient.

The present invention also is directed to oral dosage forms andpharmaceutical formulations for administration to mammals suffering fromor at risk for diseases having the characteristics of Type 1 diabetes.It will be understood that any statistically significant attenuation inthe disease symptoms of Type 1 diabetes pursuant to the treatment of thepresent invention is within the scope of the invention.

Each oral formulation according to the present invention mayadditionally comprise inert constituents including pharmaceuticallyacceptable carriers, diluents, fillers, solubilizing or emulsifyingagents and salts as is well-known in the art. For example, tablets maybe formulated in accordance with conventional procedures employing solidcarriers well-known in the art. Capsules employed in the presentinvention may be made from any pharmaceutically acceptable material suchas gelatin or cellulose derivatives. Sustained release oral deliverysystems and/or enteric coatings for orally administered dosage forms arealso contemplated such as those described in U.S. Pat. No. 4,704,295issued Nov. 3, 1987, U.S. Pat. No. 4,556,552 issued Dec. 3, 1985, U.S.Pat. No. 4,309,404 issued Jan. 5, 1982 and U.S. Pat. No. 4,309,406issued Jan. 5, 1982.

Examples of solid carriers include bentonite, silica and other commonlyused carriers. Further non-limiting examples of carriers and diluentswhich may be used in the formulations of the present invention includesaline and any physiologically buffered saline solution such asphosphate buffered saline (PBS) and water.

It will be appreciated that the unit content of active ingredient oringredients contained in an individual dose of each dosage form need notin itself constitute an effective amount since the necessary effectiveamount can be reached by administration of a plurality of dosage units.

The preferred route of administration of the dosage forms of the presentinvention is orally or enterally. Preferred oral or enteralpharmaceutical formulations or dosage forms may comprise for example,between about 1 mg and about 1000 mg of insulin.

In an alternative preferred embodiment of the present invention thepharmaceutical formulations or dosage forms of the present invention canalso be administered to mammals suffering from diseases having thecharacteristics of Type 1 diabetes in aerosol form. It is expected thatlower amounts of insulin, disease suppressive fragments or analogsthereof will be required using aerosol administration for treating orpreventing Type 1 diabetes as has been found when treating experimentalallergic encephalomyelitis (EAE) with myelin basic protein (MBP) andadjuvant arthritis with collagen as disclosed in co-pending U.S. patentapplication Ser. No. 454,806 filed Dec. 20, 1989, now abandoned. Theamounts of insulin or disease suppressive fragments or analogs thereofwhich may be administered in an aerosol dosage form would be betweenabout 0.1 mg and 10 mg per kg body weight of a mammal per day and may beadministered in single dosage form or multiple dosage forms. The exactamount to be administered will vary depending on the state and severityof a patient's disease and the physical condition of the patient.

The aerosol pharmaceutical formulations of the present invention mayinclude, as optional ingredients, pharmaceutically acceptable carriers,diluents, solubilizing or emulsifying agents, and salts of the type thatare well-known in the art. Examples of such substances include normalsaline solutions, such as physiologically buffered saline solutions, andwater.

The route of administration of insulin or disease suppressive fragmentsor analogs thereof according to this alternate embodiment of the presentinvention is in an aerosol or inhaled form. The insulin and relatedcompounds of the present invention can be administered as a dry powderor in an aqueous solution. Preferred aerosol pharmaceutical formulationsmay comprise for example, a physiologically-acceptable buffered salinesolution containing between about 1 mg and about 1000 mg of insulin,disease suppressive fragments or analogs thereof.

Dry aerosol in the form of finely divided solid particles of insulin,disease suppressive fragments or analogs thereof that are not dissolvedor suspended in a liquid are also useful in the practice of the presentinvention. The insulin may be in the form of dusting powders andcomprise finely divided particles having an average particle size ofbetween about 1 and 5 microns, preferably between 2 and 3 microns.Finely divided particles may be prepared by pulverization and screenfiltration using techniques well known in the art. The particles may beadministered by inhaling a predetermined quantity of the finely dividedmaterial, which can be in the form of a powder.

Specific non-limiting examples of the carriers and/or diluents that areuseful in the aerosol pharmaceutical formulations of the presentinvention include water and physiologically-acceptable buffered salinesolutions such as phosphate buffered saline solutions pH 7.0-8.0.

The pharmaceutical formulations of the present invention may beadministered in the form of an aerosol spray using for example, anebulizer such as those described in U.S. Pat. No. 4,624,251 issued Nov.25, 1986; U.S. Pat. No. 3,703,173 issued Nov. 21, 1972; U.S. Pat. No.3,561,444 issued Feb. 9, 1971 and U.S. Pat. No. 4,635,627 issued Jan.13, 1971. The aerosol material is inhaled by the subject to be treated.

Other systems of aerosol delivery, such as the pressurized metered doesinhaler (MDI) and the dry powder inhaler as disclosed in Newman, S. P.in Aerosols and the Lung, Clarke, S. W. and Davia, D. eds. pp. 197-224,Butterworths, London, England, 1984, can be used when practicing thepresent invention.

Aerosol delivery system of the type disclosed herein are available fromnumerous commercial sources including Fisons Corporation (Bedford,Mass.), Schering Corp. (Kenilworth, N.J.) and American Pharmoseal Co.,(Valencia, Calif.).

It is expected that the methods of the present invention areparticularly well-suited for use in pediatric or adolescent patientsdeveloping Type 1 diabetes or the first year of onset of hyperglycemiain whom the secondary effects of the disease (such as vascular damage,kidney damage and diabetic retinopathy) have not been totallymanifested. These are also the patients in whom the traumatic effects ofdaily insulin injections are usually most severely felt.

As shown in the examples presented below, oral administration of insulinto NOD mice reduced the numbers of these mice which subsequently went onto develop diabetes at all dosages tested. In addition, at the highestdose used (1 mg), none of the mice went on to develop diabetes. Inaddition, as shown in Example 2, the effects of oral insulin were notdue to a reduction in the serum glucose concentrations in these animalsand was thus not a metabolic effect. In addition, as shown in Example 3below, feeding NOD mice pancreatic extract also led to a diminution inthe number of animals which developed diabetes although to a lesserextent than those that received oral insulin.

Preliminary experiments have shown that NOD mice fed 1 mg of insulin hadfewer inflammatory immune system cells surrounding the pancreas (acondition known as insulitis) than PBS-fed NOD mice (data not shown).Therefore, oral administration of insulin to NOD mice appeared to beaffecting the infiltration of immune system cells into the pancreas andthus halting disease progression by preserving beta cell function.

The present invention is described further below in working exampleswhich are intended to illustrate the present invention without limitingits scope.

EXAMPLE 1: EFFECT OF FEEDING PORCINE-INSULIN ON SPONTANEOUS DIABETES INNOD MICE

NOD (non-obese diabetic mice) were obtained from Taconic Laboratories(Germantown, Mass.) at 4 weeks of age. There were a total of 30 animalsin each of the groups mentioned below at the beginning of theexperiment. At 41/2 weeks of age, animals were orally administeredPorcine insulin (Novo Laboratories, Danbury, Conn.). Each animalreceived two treatments orally twice a week until 9 weeks of age. Themice were fed either saline (control) or 10 micrograms, 100 micrograms,or 1 milligram of Porcine insulin. The animals were fed (gavaged) withan 18 gauge ball-point needle (Popper and Sons, Inc., New Hyde Park,N.Y.). Beginning at 10 weeks the animals were then fed once a week for atotal of 33 weeks. Beginning at 12 weeks the urine was tested weekly forthe presence of glucose using Glucosuria test tape (Eli Lilly,Indianapolis, Ind.). If an animal tested 3+ or higher on the glucoseurine test, a serum sample was taken and blood glucose measured using aglucose analyzer (Beckman). If the blood glucose was 220 or higher, theanimals were classified as diabetic and then sacrificed.

As shown in FIG. 1, no diabetes developed in any of the animals fed 1 mginsulin whereas diabetes developed in the control group and in the 10microgram and 100 microgram groups in a dose response fashion. The pvalue comparing the 1 mg fed to the control is p<0.008 whereas in the100 microgram fed, p<0.09.

The normal blood insulin level in an animal is between 120 to 170mg/decaliter or mg/dl. The blood sugars in the animals that were fed PBSthat developed diabetes were: 575, 485, 459, 500, 375, 400, 362, 395,480 mg/dl. The blood sugar in the animals that were fed 10 micrograms ofinsulin which developed diabetes were: 330, 420, 315, 459, 520, 487mg/dl. Blood sugars in animals fed 100 micrograms of insulin that becamediabetic were: 290, 600, 450, 500 mg/dl. Animals that were fed 1 mg ofinsulin were also tested in terms of blood sugar. None of the animalsdeveloped diabetes as measured by urine testing but the blood sugar wastested as a confirmation. Blood sugar on 10 representative animals fromthe group fed 1 mg insulin were: 174, 128, 125, 125, 145, 123, 136, 155,115, 130 mg/dl.

EXAMPLE 2: GLUCOSE POST ORAL INSULIN IN FASTING 7 WEEK NOD MICE

In order to determine whether any endocrinologic (i.e. direct loweringof blood sugar) effects occurred following the feeding of insulin, 7week old NOD mice (5 mice per group) were fed 1 mg of porcine insulin or1 mg of myelin basic protein (obtained from Pel Freez, Rogers, Ark.) asa control protein. Blood glucose was then measured over the next 24 hourperiod. As a positive control, a separate group of animals were injectedsubcutaneously with 20 micrograms of Porcine insulin. All animals werekept in the fasting state.

As shown in FIG. 2, animals given subcutaneous insulin had an immediatedrop in the glucose level with a decrease of approximately 80 mg % thatpersisted over a 4 hour period and then slowly returned to normal.Animals which received oral insulin or oral myelin basic protein had nodrop in the blood glucose; in fact there was an increase in bloodglucose most probably related to the protein load. Over the next 24hours there was a gradual decrease in blood glucose in all groups ofanimals since the animals were in a fasting state. This was seen both inanimals fed insulin and animals fed myelin basic protein.

These results demonstrate that there were no endocrinologic (metabolic)effects from feeding oral insulin. Given the published data in terms ofgenerating immunologic tolerance and immunologic effects following theoral administration of proteins these results strongly suggest that oraladministration of insulin affects the immunologic pathogenesis ofdiabetes in the NOD mice.

EXAMPLE 3: EFFECT OF ORAL ADMINISTRATION OF PANCREATIC EXTRACT GIVEN ATWEEKLY INTERVALS

An identical experiment to Example 1 was performed in which NOD mice(14-17 per group) were fed one homogenized pancreas derived from Lewisrats in 0.5 mls PBS. As shown in FIG. 3, there was a decrease in thedevelopment of diabetes in extract-fed animals as compared to controls.Nonetheless, the effect is not as dramatic and complete as that seen inthe animals fed insulin. These results show some effect of feedingpancreatic tissue and highlight the potency of the insulin experimentsdescribed above.

EXAMPLE 4: EFFECT OF AEROSOL ADMINISTRATION OF INSULIN ON DIABETES INNOD MICE

Insulin (0 to 1 mg as in Examples 1 and 2) will be administered to NODmice as described above in Examples 1 and 2 except in aerosol form bynebulizer.

It is expected that aerosolized insulin will be effective in preventingthe manifestation of symptoms (i.e. hyperglycemia) of Type 1 diabetes inthe treated mice.

EXAMPLE 5: TREATMENT OF INITIAL STAGES OF DIABETES IN NOD MICE

Insulin (0 to 1 mg as in Examples 1 and 2) will be administered to NODmice which are suffering from hyperglycemia in an oral and aerosol form.The treatments will begin by weeks 12-20 of age, by which the serumglucose levels are at hyperglycemic concentrations in a certainpercentage of animals prior to onset of diabetes.

It is expected that both oral and aerosol administration of insulin willlead to reduction in serum glucose concentrations to near normal levelsand preservation of beta cell function.

What is claimed is:
 1. A method for suppressing autoimmune responseagainst pancreatic beta cells in a mammal comprising orally or enterallyadministering to said mammal an mount of a composition comprisinginsulin effective to suppress said autoimmune response without causing adecrease in the blood sugar level of said mammal within 4 hours aftersaid administration.
 2. The method of claim 1 wherein saidadministration is via the oral route.
 3. The method of claim 2 whereinsaid administration suppresses autoimmune destruction of pancreatic betacells.
 4. The method of claim 1 wherein said composition is orallyadministered as an aqueous suspension or solution of insulin.
 5. Themethod of claim 1 wherein said composition is orally administered in aform selected from the group consisting of a tablet, a capsule and acaplet.
 6. The method of claim 5 wherein said form comprises apharmaceutically acceptable carrier or diluent.
 7. The method of claim 1wherein said composition consists of insulin.
 8. The method of claim 1,wherein the administration continues in single or multiple doses for atleast 12 weeks.
 9. The method of claim 1 wherein said mammal suffersfrom a disease selected from the group consisting of Type 1 diabetes andanimal disease states that serve as models for Type 1 diabetes.
 10. Amethod for treating a mammal suffering from a disease selected from thegroup consisting of Type 1 diabetes and animal disease states that serveas models for Type 1 diabetes comprising the step of orally or enterallyadministering to said mammal a composition containing an amount ofinsulin, effective to suppress an autoimmune response associated withsaid disease, without causing a decrease in blood sugar level of saidmammal within 4 hours after said administration.
 11. A method forpreventing or suppressing the onset of Type 1 diabetes in a mammal bysuppressing an autoimmune response leading to Type 1 diabetes,comprising the step of orally or enterally administering to said mammalan effective amount of a composition comprising insulin, prior to saidonset and without causing a decrease in the blood sugar level of saidmammal within 4 hours after said administration.
 12. The method of claim11 wherein said administration is oral.
 13. The method of claim 11wherein said composition consists of insulin.
 14. A method forsuppressing autoimmune reaction against pancreatic beta cells in apatient comprising orally or enterally administering to said patient aneffective amount for suppressing said autoimmune reaction of acomposition comprising insulin, said composition not causing a decreasein the blood sugar level of said patient within 4 hours after saidadministration.
 15. A method of treating a human patient suffering froma stage of Type 1 diabetes characterized by an ongoing autoimmuneresponse comprising orally administering to said patient an effectiveamount of a composition comprising insulin that wherein the insulin issusceptible to degradation by proteolytic enzymes in the digestivetract, said composition suppressing said ongoing autoimmune responsewithout causing a decrease in the blood sugar level of said patientwithin 4 hours afar said administration.
 16. A method for preventing orsuppressing the onset of Type 1 diabetes in a human patient comprisingorally administering to said patient an effective amount for suppressingautoimmune response associated with Type 1 diabetes of a compositioncomprising insulin that wherein the insulin is susceptible todegradation by proteolytic enzymes in the digestive tract, saidcomposition not causing a decrease in the blood sugar level of saidpatient within 4 hours after said administration.
 17. A method oftreating a human patient suffering from a state of Type 1 diabetescharacterized by an autoimmune response, comprising oral administrationto said patient of a composition comprising insulin in amount effectiveto produce at least one physiological response selected from the groupconsisting of suppressing said autoimmune response, reducing destructionof beta cells, and eliciting suppressor T-cells that recognize insulin,said composition not causing a decrease in the blood sugar level of saidpatient within 4 hours after said administration.
 18. The method ofclaim 17, wherein the administration continues in single or multipledoses for at least 12 weeks.
 19. A method for suppressing autoimmuneresponse against pancreatic beta cells in a mammal comprising orally orenterally administering to said mammal an amount of a compositioncomprising insulin effective to suppress said autoimmune response saidcomposition not causing an accompanying decrease in the blood sugarlevel of said mammal.